CN1069099C - Novel anthraeycline compound derivatives and its medical preparation - Google Patents

Abstract

The present invention discloses dimers, trimers or tetramers of anthracycline compounds, which can be directly chemically combined with anthracycline compounds with anticancer activity, and a high molecular block copolymer prepared by treating with alkali -Drug complex preparation, wherein the polymer block copolymer having a hydrophilic polymer segment and a hydrophobic polymer segment forms a micelle with the hydrophilic segment as the outer layer, and contains an anthracycline dimer in its hydrophobic core , trimer or tetramer, and other drugs if necessary. The above-mentioned pharmaceutical preparation has high drug efficacy and low toxicity.

Description

Dimers and trimers of doxorubicin

field of invention

The present invention relates to a novel anthracycline compound derivative and a high molecular block copolymer-drug compound preparation containing the derivative.

Background of the invention

Daunorubicin (U.K. Patent No. 1003383, U.S. Patent No. 3616242), Adriamycin (U.S. Patent No. 3,590,028, U.S. Patent No. 3,803,124) obtained from the culture solution of actinomycetes are known as anthracycline anticancer agents. They have broad-spectrum anti-tumor properties against experimental tumors and are also widely used as cancer chemotherapeutics. On the other hand, however, they are known to often cause severe side effects such as leukopenia, alopecia, cardiomyopathy and the like.

To solve this problem, various derivatives have been proposed. For example, pirarubicin (generic name) attempts to reduce its toxicity by introducing a tetrahydropyranyl group at the 4' position of the doxorubicin sugar moiety.

Likewise, epirubicin (common name) is a compound in which the hydroxyl group at the 4' position of the doxorubicin sugar moiety is attached to the alpha position in an attempt to reduce its toxicity.

However, although these drugs have lower toxicity compared with doxorubicin, their problems, such as limited total dose, etc., have not been completely solved.

On the other hand, improving the solubility of slightly water-soluble drugs using high molecular weight micelles formed by block copolymers is a known technique. It has been proven in Japanese Patent Application (Open) 2-300133, Japanese Patent Application (Open) 6-107565, Japanese Patent Application (Open) 5-955, Japanese Patent Application (Open) 5-124969, Japanese Patent Application (Open) 5-117385, Japanese Patent Application (Open) 6-206830, Japanese Patent Application (Open) 7-69900 and Japanese Patent Application (Open) 6-206815, the polymer block copolymer drug compound preparation has more than Adriamycin better anticancer effect.

disclosure of invention

The inventors of the present invention have conducted in-depth research on the polymer block copolymer-drug compound preparation which has higher drug efficacy and lower toxicity than the existing polymer block copolymer-drug compound preparation. As a result, the present invention has been accomplished.

Therefore, the present invention relates to:

(1) dimers, trimers or tetramers of anthracycline compounds, which can be prepared by directly chemically bonding the anthracycline compounds with anticancer activity to each other through alkali treatment;

(2) The dimer, trimer or tetramer of the anthracycline compound described in the above item (1), wherein the anthracycline compound contains at least A compound of star, epirubicin and its acid salt;

(3) dimers of anthracyclines, which can be directly chemically bonded to doxorubicin molecules or their acid salts by alkali treatment or make doxorubicin or its acid salts and daunorubicin It is obtained by direct chemical bonding of its acid salt;

(4) The dimer, trimer or tetramer of the anthracycline compound described in the above item (1), (2) or (3), wherein the mutual bonding mode of the anthracycline compound is Schiff Base-bonding mode;

(5) Adriamycin dimers having the following structural formula (AA):

(6) A doxorubicin trimer prepared by directly chemically bonding doxorubicin molecules or their acid salts to each other by alkali treatment and having a mass spectrum as shown in Figure 7;

(7) A polymer block copolymer-drug compound preparation, wherein the polymer block copolymer having a hydrophilic polymer segment and a hydrophobic polymer segment forms a micelle in which the hydrophilic segment is the outer layer, and in its hydrophobic inner layer The core contains dimers, trimers or tetramers of anthracyclines and, if necessary, other drugs;

(8) The polymer block copolymer-drug composite preparation as described in the above item (7), wherein the dimer, trimer or tetramer of anthracycline compound is the above item (1), (2) ), (3), (4), (5) or (6) dimers, trimers or tetramers of anthracycline compounds described in item;

(9) The high molecular block copolymer-drug compound preparation as described in the above item (7) or (8), wherein the high molecular block copolymer has the structure of the following general formula (1) or (2):

In the formula, _R1 represents a hydrogen atom or a lower alkyl group, _R2 represents a linking group, _R3 represents a methylene group or an ethylene group, _R4 independently represents a hydroxyl group or an anthracycline compound residue with anticancer activity, R ₅ represents a hydrogen atom or a protecting group, n is an integer between 5-1000, m is an integer between 2-300, and x is an integer between 0-300, provided that x is not greater than m;

(10) The polymer block copolymer-drug composite preparation as described in the above item (9), wherein the anthracycline compound residue having anticancer activity is a group represented by the following general formula (3):

In the formula, Y represents -CH ₂ OH or -CH ₃ , and Z represents H or

(11) The polymer block copolymer-drug composite preparation as described in the above item (7), (8), (9) or (10), wherein in the micelles formed by the polymer block copolymer The core contains 2-60% by weight of dimers, trimers or tetramers of anthracycline compounds, based on high-molecular block copolymers;

(12) The polymer block copolymer-drug compound preparation as described in the above item (7), (8), (9), (10) or (11), wherein the anthracycline compound dimer, tri The polymer or tetramer is a dimer of doxorubicin;

(13) The polymer block copolymer-drug compound preparation as described in the above item (7), (8), (9), (10), (11) or (12), wherein The micelle inner core formed by the block copolymer contains dimers, trimers or tetramers of anthracycline anticancer drugs and anthracycline compounds at a weight ratio of 1:0.5 to 20;

(14) The polymer block copolymer-drug composite preparation as described in the above item (13), wherein the weight ratio of the dimer, trimer or tetramer of the anthracycline anticancer drug to the anthracycline compound is 1:0.7～10;

(15) The polymer block copolymer-drug composite preparation as described in the above item (13), which contains a dimer of an anthracycline anticancer drug and an anthracycline compound in a weight ratio of 1:1 to 5 , trimer or tetramer;

(16) The polymer block copolymer-drug compound preparation as described in the above item (13), (14) or (15), wherein the anthracycline anticancer drug is at least selected from doxorubicin, daunorubicin A medicine of methazine, pirarubicin, epirubicin and their acid salts;

(17) A polymer block copolymer-drug composite preparation, wherein the polymer block copolymer having a hydrophilic polymer segment and a hydrophobic polymer segment forms a micelle in which the hydrophilic segment is the outer layer, and the hydrophobic The inner core contains an anthracycline anticancer drug, which is characterized in that when the drug preparation is intravenously injected into CDF1 mice aged 7 to 9 weeks, if the amount of the anthracycline anticancer drug in the injection preparation is set as 100, then 1 The content of anthracycline anticancer drugs in 1 milliliter of rat plasma after 1 hour is 10 (% dose/ml) or more;

(18) The pharmaceutical preparation as described in the above item (17), wherein the content of anthracycline anticancer drugs in 1 ml of mouse plasma 1 hour after injection is 20-60 (% dose/ml);

(19) The pharmaceutical preparation as described in the above item (17) or (18), wherein at least one anthracycline anticancer drug is selected from the group consisting of adriamycin, daunorubicin, pirarubicin, epirubicin, Star and its acid salts;

(20) The pharmaceutical preparation according to any one of the above items (7) to (19), which is characterized in that it is used for the treatment of solid cancer.

Introduction to the drawings

Fig. 1 is the infrared absorption spectrogram of doxorubicin dimer.

Fig. 2 is the ultraviolet spectrogram of doxorubicin dimer.

Figure 3 is the mass spectrum of doxorubicin dimer.

Fig. 4 is an infrared absorption spectrum of a compound possibly having the structure of formula (4), which is a compound produced together with doxorubicin when doxorubicin dimer is treated with acid.

Fig. 5 is the ultraviolet spectrogram of the compound possibly having the structure of formula (4), which is a compound produced together with doxorubicin when doxorubicin dimer is treated with acid.

Fig. 6 is a mass chromatogram (m/z=560) of a compound possibly having the structure of formula (4), which is a compound produced together with doxorubicin when doxorubicin dimer is treated with acid.

Figure 7 is the mass spectrum of doxorubicin trimer.

Fig. 8, 9, 10 or 11 are ^{the 1} H-dimensional spectrum, ¹³ C-dimensional spectrum, COZY spectrum or CH COZY spectrum respectively obtained from the NMR analysis of the components separated in Example 1(2).

Figure 12 is the mass spectrogram of the dimer of daunorubicin and doxorubicin.

Figure 13 is a high pressure liquid chromatogram of the pharmaceutical preparation prepared in Example 3.

14 is a high pressure liquid chromatogram of the pharmaceutical preparation prepared in Example 4.

Figure 15 is a high pressure liquid chromatogram of the pharmaceutical preparation prepared in Example 5.

Fig. 16 is a graph showing the tumor growth curve of colon (Colon) 26 adenocarcinoma in mice administered with doxorubicin hydrochloride in application example 1.

Fig. 17 is a tumor growth curve of colon (Colon) 26 adenocarcinoma in mice administered with the pharmaceutical preparation prepared in Example 3 in Application Example 1.

Figure 18 is a high pressure liquid chromatogram of the pharmaceutical preparation prepared in Example 6.

Figure 19 is a high pressure liquid chromatogram of the pharmaceutical preparation prepared in Example 7.

Figure 20 is a high pressure liquid chromatogram of the pharmaceutical preparation prepared in Example 8.

Figure 21 is a high pressure liquid chromatogram of the pharmaceutical preparation prepared in Example 9.

Figure 22 is a high pressure liquid chromatogram of the pharmaceutical preparation prepared in Example 10.

Figure 23 is a high pressure liquid chromatogram of the pharmaceutical preparation prepared in Example 11.

Figure 24 is a high pressure liquid chromatogram of the pharmaceutical preparation prepared in Example 12.

Figure 25, 26 or 27 is the tumor of mouse colon (Colon) 26 adenocarcinoma after administration of doxorubicin hydrochloride, the pharmaceutical preparation prepared in Example 8 or the pharmaceutical preparation prepared in Example 12 to mice in Application Example 3 respectively Growth graph.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is described in detail below.

According to the present invention, a pharmaceutical preparation with higher efficacy and lower toxicity than conventional anthracycline anticancer drugs or existing polymer block copolymer-drug composite preparations can be obtained.

Examples of anthracycline compounds having anticancer activity used in the present invention include doxorubicin, daunorubicin, pirarubicin, epirubicin, and acid salts thereof.

The method for preparing the dimer, trimer or tetramer of the anthracycline compound of the present invention is not particularly limited, for example, the dimer, trimer or tetramer can be obtained by treating the anthracycline compound with anticancer activity with alkali polymers or tetramers. During alkali treatment, dimers and trimers of anthracyclines can be obtained through mutual direct chemical bonding (that is, not chemical bonding with a crosslinking agent, but chemical bonding formed by the reaction between functional groups of anthracyclines) compounds or tetramers.

Dimers, trimers or tetramers of anthracyclines can be prepared by bonding molecules of the same or different compounds.

An example of the base treatment is a method in which an anthracycline compound is dissolved in a solvent, and then a base is added. The solvent used is not particularly limited as long as it can dissolve the compound. Examples thereof include water, N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), dioxane, tetrahydrofuran (THF), methanol, acetonitrile, and mixed solvents thereof.

If the added base can be dissolved in the solvent, and the solution after adding the base has a pH value of 7-14, then there is no limit to the added base, which can be any inorganic base, organic base and salts thereof. The concentration of the base is also not particularly limited. Examples of desired bases include sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium phosphate, dipotassium hydrogen phosphate, phosphoric acid Potassium dihydrogen, secondary or tertiary amines having 2 to 20 carbon atoms, and acid addition salts thereof. During alkali treatment, the pH value is 7-14, preferably 8-10.

If the solution does not freeze or boil, the temperature of the alkali treatment is not particularly limited, but is preferably 0-50°C, more preferably 0-40°C. The treatment time is from 1 minute to 120 hours, preferably from 10 minutes to 24 hours.

The resulting dimers, trimers or tetramers of anthracyclines can be purified using known purification techniques. For example, solid matter can be obtained by lyophilization, precipitation, etc., or by exchanging solvents by dialysis or ultrafiltration followed by lyophilization, precipitation, and the like. When it is necessary to further purify the obtained solid substance, thin layer chromatography, liquid chromatography or the like can be used.

When a compound having both a carbonyl structure substituent and an amino group is used as an anthracycline compound, or a compound having a carbonyl structure substituent is used in combination with another compound having an amino group, the anthracycline is obtained by the above-mentioned base treatment Compounds are dimers, trimers or tetramers that are chemically combined with each other through Schiff base bonds. As a result, it is satisfactory to use a compound having both a carbonyl structural substituent and an amino group, or a compound having a carbonyl structural substituent in combination with a compound having an amino group, as an anthracycline compound. In this regard, examples of substituents having a carbonyl structure include acyl groups having 2 to 5 carbon atoms which may be substituted by hydroxyl or halogen atoms (such as fluorine, chlorine, bromine, and iodine atoms); or acyl groups which may be substituted by hydroxyl or halogen atoms Acylalkyl having 3-10 carbon atoms.

When dimers, trimers or tetramers formed by bonding anthracyclines to each other via Schiff base bonds are treated with an acid, at least one anthracycline used as a raw material is produced. An example of the acid treatment includes a method of dissolving dimers, trimers or tetramers of anthracycline compounds in a solvent and adding an acid. The solvent used is not particularly limited as long as it can dissolve the compound. Examples of solvents include water, N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), dioxane, tetrahydrofuran (THF), methanol, acetonitrile, and mixed solvents thereof. Any of inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid and phosphoric acid or organic acids such as formic acid, acetic acid and trifluoroacetic acid can be used as the acid to be added.

The pH value is preferably 2-4 during acid treatment. The temperature of the acid treatment is also not particularly limited if the solution does not freeze or boil, but is preferably 0-50°C, more preferably 20-40°C. The treatment time is from 1 minute to 120 hours, more preferably from 24 to 72 hours.

Doxorubicin dimers having the infrared absorption spectrum shown in FIG. 1 and the ultraviolet spectrum shown in FIG. 2 can be cited as the anthracyclines of the present invention that can be directly chemically bonded to the anthracyclines by the above-mentioned alkali treatment. Compound dimers, trimers or tetramers. This doxorubicin dimer also has the mass spectrum shown in FIG. 3 . This doxorubicin dimer has the structure represented by the above structural formula (AA).

Infrared absorption spectrum: 1676, 1417, 1217, 1158cm ^-1 ;

UV spectrum: λmas=486nm;

Mass spectrum (ESI), m/z(%): 1067(100), 964(10), 938(15), 921(13), 653(20), 524(20), 506(50), 488(98 ).

In this regard, the instruments and measurement conditions used to measure these spectra are as follows. The infrared absorption spectrum was measured with System 2000 manufactured by Perkin-Elmer Co. and the potassium bromide pellet method. The ultraviolet spectrum was measured in a methanol solution with a spectrophotometer model U3200 manufactured by Hitachi Co. . The mass spectrum was measured with a QUATTRO2 mass spectrometer manufactured by VG Company and electrospray method.

When treated with acid, the doxorubicin dimer produces doxorubicin and possibly compounds of the following formula (4):

The infrared absorption spectrum, ultraviolet spectrum or mass chromatogram (m/z=560, obtained with liquid chromatography/mass spectrometry (LC/MS)) of the compound that may have the general formula (4) structure are shown in Fig. 4, Fig. 5 or Fig. 6 in.

In this regard, the instruments and measurement conditions used to measure these spectra and chromatograms are as follows. Infrared absorption spectra were measured with the same apparatus and under the same conditions as those used to measure the spectra shown in FIG. 1 . The ultraviolet spectrum was measured in a benzyl alcohol solution with a model U3200 manufactured by Hitachi Corporation. Instruments and measurement conditions for measuring mass chromatograms by LC/MS are as follows.

LC:

Column: C4 300 Å/5 µm, manufactured by Waters Co.;

Eluent: acetonitrile/0.1% trifluoroacetic acid+0.05% MS7 (MS7; manufactured by Gasukuro Kogyo Co.);

Gradient elution:

Time (min) 0 20 25 30 35 36 40

Acetonitrile concentration (%) 22 40 50 90 90 22 22

Flow rate: 1ml/min

MS: QUATTRO2 (electrospray method) manufactured by VG Corporation.

The doxorubicin trimer having the mass spectrum shown in Fig. 7 and capable of producing doxorubicin when treated with acid can be cited as another alternative of the present invention that can be directly chemically combined with anthracyclines by the above-mentioned base treatment method. Examples of obtained dimers, trimers or tetramers of anthracyclines.

The structure of the hydrophilic polymer segment of the polymer block copolymer used in the polymer block copolymer-drug compound preparation of the present invention can include polyethylene glycol, polysaccharide, polyacrylamide, polymethacrylamide, polyethylene The structure of alcohol, polyvinylpyrrolidone, chitosan, etc. is not particularly limited as long as it has a structure of a hydrophilic polymer. A particularly preferred structure is the polyethylene glycol structure.

The structure of the hydrophobic polymer segment can include polystyrene, polyamino acid (polyaspartic acid, polyglutamic acid, polylysine, etc.), polyacrylic acid, polymethacrylic acid, polymaleic acid, or derivatives thereof The structure of the compound or its salt is not particularly limited as long as it has the structure of a hydrophobic polymer. Among them, polyamino acids, derivatives thereof, or salts thereof are preferable, and polyaspartic acid, polyglutamic acid, derivatives thereof, or salts thereof are particularly preferable. Although not particularly limited, examples of the salt include sodium salt, potassium salt and the like.

Examples of derivatives of the polyamino acid structure include derivatives in which hydrophobic compounds such as aromatic amines, aliphatic amines, aromatic alcohols, aliphatic alcohols, aromatic thiols, and aliphatic thiols are attached to their side chains, as long as The hydrophobic group can be connected to the side chain and make the polyamino acid segment hydrophobic, and there is no special limitation on the hydrophobic group connected to the side chain. Preferred derivatives are polyaspartic acid or polyglutamic acid derivatives in which an amine having an aromatic ring is attached to a side chain.

Preferable examples of the high-molecular block copolymer include high-molecular block copolymers having the above-mentioned chemical structure represented by the general formula (1) or (2).

In the above general formula (1) or (2), R ₁ represents hydrogen or a lower alkyl group, wherein an example of the lower alkyl group is an alkyl group having 1 to 3 carbon atoms, preferably a methyl group.

The linking group represented by _R2 has a structure corresponding to the method and compound used to form the polyamino acid fragment at the end of the polyethylene glycol fragment, so that the end of the compound forming the polyethylene glycol fragment is converted into a compound suitable for the above-mentioned formation process. structure. Its examples include alkylene groups having 1 to 8 carbon atoms, such as methylene, ethylene, 1,2-propylene, 1,3-propylene and isobutylene, wherein 1,3 - Propylene is preferred.

R ₃ represents methylene, ethylene, preferably methylene.

R ₄ independently represents a hydroxyl group or a residue of an anthracycline compound having anticancer activity. Various residues can be used as the residue of the anthracycline compound having anticancer activity, and there is no particular limitation, but a group represented by the general formula (3) can be preferably used. Illustrative examples of the group represented by the general formula (3) include adriamycin residues, daunorubicin residues, pirarubicin residues, and epirubicin residues.

Although R ₄ independently represents a hydroxyl group or an anthracycline compound residue with anticancer activity, at least a part, preferably 5-80% of all R ₄ groups present in the polymer block copolymer are preferably having The anthracycline compound residues with anticancer activity, more preferably 20-60% of the R ₄ groups present in the polymer block copolymer are the above anthracycline compound residues.

Although R _{independently} represents a hydroxyl group or a residue of an anthracycline compound having anticancer activity, a group having an anthracene skeleton or an anthraquinone skeleton has an anthracene skeleton as disclosed in Japanese Patent Application (Kokai) 6-206830 Or a substituent of the anthraquinone skeleton can be used to replace the residue of the above-mentioned anthracycline compound.

R ₅ represents hydrogen or a protecting group, wherein an aliphatic or aromatic acyl group can be cited as a protecting group. Although not particularly limited, the protecting group can be introduced by a known method, such as a method using an acid anhydride or a method using an acid halide. A hydrogen atom or an acetyl group is suitable as R ₅ .

In addition, n is 5-1000, preferably 15-400, m is 2-300, preferably 10-100, x is 0-300, preferably 0-100.

The molecular weight of the high-molecular block copolymer is preferably 1,000-100,000, more preferably 5,000-50,000, although the molecular weight is not particularly limited when the block copolymer is soluble in water. As long as the pharmaceutical preparation of the present invention can maintain water solubility, the ratio of the hydrophilic polymer segment to the hydrophobic polymer segment in the polymer block copolymer is not particularly limited, but it is preferably 1: 0.1 to 10 (by weight), more preferably Preferably, it is 1:0.1 to 5 (by weight).

Although drugs other than dimers, trimers, or tetramers of anthracyclines that may be included in the core of high molecular weight block copolymer micelles are not necessarily essential components, examples thereof include doxorubicin , daunorubicin, pirarubicin, epirubicin, methotrexate, mitomycin, etoposide, cisplatin and its derivatives and other anticancer drugs. Among them, anthracycline anticancer drugs are preferred, and adriamycin, daunorubicin, pirarubicin, epirubicin or their acid salts are particularly preferred.

The content of the dimer, trimer or tetramer of the anthracycline compound in the polymer block copolymer-drug composite preparation is preferably 1-100% by weight, more preferably 2-60% by weight (based on polymer based on block copolymers). However, as long as the micelle-forming ability of the high-molecular block copolymer-drug composite preparation is not impaired, there is no problem in using the dimer, trimer or tetramer in as large an amount as possible.

The content of other drugs other than anthracycline compound dimers, trimers or tetramers in the polymer block copolymer-drug composite preparation is preferably 0-100% by weight, more preferably 2-60% by weight, Based on polymer block copolymers. However, as long as the micelle-forming ability of the polymer block copolymer-drug composite preparation is not impaired, there is no problem in using as large an amount of the other drug as possible.

When the polymer block copolymer-drug compound preparation contains "drugs other than dimers, trimers or tetramers of anthracycline compounds", "dimers, trimers or tetramers of anthracycline compounds The weight ratio of "drugs other than polymers" to "dimers, trimers or tetramers of anthracycline compounds" is generally 1:0.05-100, preferably 1:0.5-20, more preferably 1: 0.7-10, preferably 1: 1-5.

Although there is no particular limitation on the dimer, trimer or tetramer of the anthracycline compound contained in the core of the polymer block copolymer micelle, the above-mentioned items (1) to (6) Dimers, trimers or tetramers of anthracyclines are suitable. The micelle core may contain only one of these dimers, trimers or tetramers, or the micelle core may contain two or more of these dimers, trimers or tetramers. Various.

Methods for preparing high molecular block copolymers are known, for example, it can be prepared by making the compounds (such as polyethylene glycol, polysaccharide, polyacrylamide, polymethacrylamide) constituting the hydrophilic polymer segment , polyvinyl alcohol, polyvinylpyrrolidone, chitosan, etc., or derivatives thereof) or a terminal-modified product thereof reacts with a polymer compound constituting a hydrophobic polymer segment, or reacts a compound constituting a hydrophilic polymer segment or The terminal-modified product thereof is reacted with a polymerizable monomer, and then, if necessary, undergoes a chemical reaction such as a derivative formation reaction.

When the hydrophobic polymer segment has a polymerized carboxylic acid structure, an example of a derivative-forming reaction can be cited as a reaction with a hydrophobic compound to increase its hydrophobicity. The hydrophobic compound forms an ester bond, an amide bond, etc., and is thus attached to the high molecular block copolymer. These reactions can be carried out by conventionally known methods such as esterification and amidation. For example, when a hydrophobic compound is linked by an amide bond to a high-molecular block copolymer (raw material copolymer) having a hydrophilic polymer segment and a polymer carboxylic acid segment, the reaction can be carried out by a conventional method called a peptide bond forming method . For example, an acid halide method, an acid anhydride method, or a coupling method can be used, and among them, the coupling method using a condensing agent is suitable. As the condensing agent, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride ( EDC·HCl), dicyclohexylcarbodiimide (DCC), carbonylimidazole (CDI), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (EEDQ), diphenylphosphine Acyl azide (DPPA), etc. The amount of the condensing agent used is preferably 0.5-20 mol/mol of the hydrophobic compound, more preferably 1-10 mol/mol of the hydrophobic compound. In this case, N-hydroxysuccinimide (HONSu), 1-hydroxybenzotriazole (HOBt), N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) etc. coexist.

When carrying out the reaction of connecting the hydrophobic compound to the raw copolymer, the amount of the hydrophobic compound is not particularly limited, but its usage is generally 0.1-2 moles (based on 1 equivalent of the carboxyl group in the raw copolymer).

The condensation reaction is preferably carried out in a solvent. For example, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dioxane, tetrahydrofuran (DMF), water or a solvent mixture thereof can be used as the solvent, without particular limitation. Although not particularly limited, the solvent is generally used in an amount of 1 to 500 times the weight of the raw copolymer.

The condensation reaction is preferably carried out at a temperature of -10-50°C, more preferably -5-40°C. The reaction may be complete when the reaction proceeds for 2-48 hours.

The polymer block copolymer-drug composite preparation of the present invention can be prepared, for example, by the following method. In the first method, the obtained high molecular weight block copolymer is dissolved in a solvent. Examples of the solvent used may include N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), dioxane, tetrahydrofuran (DMF), water or a solvent mixture thereof, and the like. Among them, DMF or a mixed solvent of DMF and water is preferable. The dimer, trimer or tetramer of the anthracycline compound is added in an amount of 1-200% by weight (based on the polymer block copolymer) in the solution, and the mixture is stirred. When the solvent in the mixture solution is replaced with water by means of dialysis, ultrafiltration and the like, the desired polymer block copolymer-drug composite preparation is obtained. When other drugs are also included, they can be added together with dimers, trimers or tetramers of anthracycline compounds in an amount of 1-200% (by weight) (based on polymer block copolymers) benchmark).

In the second method, high-molecular block copolymers can also be prepared by simultaneously synthesizing dimers, trimers or tetramers of anthracyclines and incorporating them into high-molecular block copolymers- Drug compound preparations. For example, the polymer block copolymer is dissolved in a solvent, and examples of the solvent used may include N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dioxane, tetrahydrofuran (DMF ), water or its solvent mixture, etc. Among them, DMF or a mixed solvent of DMF and water is preferable. An anthracycline compound or a salt thereof (such as the above-mentioned anthracycline anticancer drug) is dissolved in the solution, and a base is added, followed by stirring. When the solvent in the mixture solution is replaced with water by means of dialysis, ultrafiltration and the like, the desired polymer block copolymer-drug composite preparation is obtained.

In the second method, the component ratio of dimers, trimers or tetramers of anthracycline compounds to other drugs in the polymer block copolymer-drug composite preparation can be controlled by the following method. For example, the component ratio of dimers, trimers or tetramers of anthracyclines and anthracyclines (anthracycline anticancer drugs) in the resulting polymer block copolymer-drug composite preparation can be changed by The amount of anthracycline compound or its salt (anthracycline anticancer drug) added (based on the polymer block copolymer), or the pH value is changed to control.

The present invention also relates to a polymer block copolymer-drug compound preparation, wherein the polymer block copolymer having a hydrophilic polymer segment and a hydrophobic polymer segment forms a micelle with the hydrophilic segment as the outer layer, and in its hydrophobic inner layer The core contains an anthracycline anticancer drug, which is characterized in that when the drug preparation is injected intravenously to CDF1 mice aged 7 to 9 weeks, if the amount of the anthracycline anticancer drug in the injection preparation is set to 100, then inject 1 After 1 hour, the content of anthracycline anticancer drugs in 1 ml of mouse plasma is 10 (% dose/ml) or more, preferably 20-60.

The above-mentioned polymer block copolymer-drug composite preparations containing dimers, trimers or tetramers of anthracycline compounds and anthracycline anticancer drugs in the micelle core can be cited as such pharmaceutical preparations .

When an existing commercially available cancer drug, such as doxorubicin, is injected intravenously into humans, its blood level decreases within a very short period of time. On the contrary, when the pharmaceutical preparation of the present invention is injected intravenously, its blood concentration is maintained at a high level for a long time, and as a result, anthracycline anticancer drugs can be entered into tumor tissues in large quantities. Therefore, cancer can be effectively treated.

When the polymer block copolymer-drug composite preparation of the present invention is used as an anticancer drug, it has high pharmacological effects. For example, it has much higher anticancer activity than doxorubicin at almost the same dose. In particular, it can show a dramatic effect on the disappearance of solid cancers. Therefore, it is particularly effective in the treatment of patients suffering from solid cancers such as lung cancer, digestive system cancer, breast cancer, bladder cancer, and osteogenic sarcoma. In addition, the polymer block copolymer-drug composite preparation of the present invention has a good effect of reducing toxicity.

The pharmaceutical preparation of the present invention can be used in different conventional dosage forms, such as solid formulation, ointment, liquid formulation and the like. These formulations can be prepared by mixing the pharmaceutical preparation of the present invention with pharmaceutically acceptable additives such as carriers, fillers, diluents, and solubilizers. And when it is used as an anticancer drug, an injection form is generally used, which preferably contains 99.99-1% of additives (based on the total formulation). Expressed by the total amount of dimers, trimers or tetramers of anthracyclines and other drugs, its dosage is about 10-200 mg/m ² /week. Give 1-3 times a week.

Example

The present invention is illustrated with reference to the following examples.

Example 1

(1) 10 mg of doxorubicin hydrochloride was dissolved in a mixed solvent consisting of 3 ml of DMF, 1 ml of water and 10 microliters of triethylamine, and reacted in the dark at 28° C. for 12 hours. Using a dialysis membrane with a nominal molecular weight cutoff (molecular weight cutoff) of 1000, the reaction solution was dialyzed against water, so that the solvent and water were exchanged. After separation and purification by HPLC, an aqueous solution of doxorubicin dimer was obtained. It was then lyophilized to obtain a solid doxorubicin dimer having a structure represented by formula (AA).

This doxorubicin dimer has the above-mentioned infrared absorption spectrum, ultraviolet spectrum and LC/MS mass spectrum. The resulting doxorubicin dimer was treated with 1% acetic acid. The mass spectrum of the obtained product is shown in FIG. 6 .

In this regard, the apparatus and conditions used for HPLC separation and purification, and measurement spectra and chromatograms are the same as previously described.

(2) 500 mg of doxorubicin hydrochloride was dissolved in a mixed solvent consisting of 40 ml of DMF and 40 ml of methanol, 1.2 ml of triethylamine was added to the solution, and allowed to react at 25° C. for 12 hours. The reaction solution was purified with a column made of 350 ml of LH-20 (manufactured by Pharmacia) packed in a glass tube with an inner diameter of 26 mm and a length of 65 cm. During column purification, methanol was used as the mobile phase. The flow rate of methanol was 5 ml/min. The eluates were collected in 5 ml portions, the 11th to 25th eluates were combined, evaporated to dryness, and analyzed by a mass spectrometer to confirm that a dimer of doxorubicin had formed. The results of high-resolution mass spectrometry showed that its molecular formula was C ₅₄ H ₅₄ N ₂ O ₂₁ .

10 mg of doxorubicin dimer thus obtained was dissolved in 2 ml of methanol, 1 mg of NaBH ₃ CN was added to this solution, allowed to react at room temperature for 12 hours, mixed with 3 ml of 1N hydrochloric acid, and then reacted for 12 Hour. The reaction solution was analyzed by LC/MS, and after separating the peak of m/z 524, it was analyzed by NMR. ^{The 1} H-dimensional spectrum obtained by NMR analysis is shown in FIG. 8 , ^{the 13} C-dimensional spectrum is shown in FIG. 9 , the COZY spectrum is shown in FIG. 10 , and the CH COZY spectrum is shown in FIG. 11 .

On the basis of these results, the compound was determined to be a dimer of doxorubicin having the structure of formula (AA).

(3) 500 mg of doxorubicin hydrochloride was dissolved in a mixed solvent consisting of 40 ml of DMF and 40 ml of methanol, 1.2 ml of triethylamine was added to the solution, and allowed to react at 25° C. for 12 hours. The reaction solution was purified with a column made of 350 ml of LH-20 (manufactured by Pharmacia) packed in a glass tube with an inner diameter of 26 mm and a length of 65 cm. Methanol was used as the mobile phase during column purification. The flow rate of methanol was 5 ml/min. The eluate was collected in 5 ml portions, the 5th to 9th eluents were combined, evaporated to dryness, and analyzed by a mass spectrometer to obtain the mass spectrum shown in Figure 7. This result confirmed the formation of doxorubicin trimer.

Example 2

5 mg of doxorubicin hydrochloride and 5 mg of daunorubicin hydrochloride were dissolved in a mixed solvent consisting of 3 ml of DMF, 1 ml of water, and 10 µl of triethylamine, and allowed to react at 28° C. in the dark for 12 hours. Without purification, the reaction solution was subjected to mass spectrometry analysis by LC/MS to confirm the formation of doxorubicin dimer and the formation of daunorubicin and doxorubicin dimer.

Among these products, doxorubicin dimer showed the same mass spectrum as above. The mass spectra of daunorubicin and doxorubicin dimers are shown in FIG. 12 . Under these reaction conditions, no dimer of daunorubicin was formed. In this regard, the apparatus and conditions used to obtain these spectra are the same as above.

Mass spectrum (ESI) of daunorubicin and doxorubicin dimer, m/z(%):1051(90),948(15),922(20),904(10),653(20),524 (30), 506 (50), 488 (100).

Example 3

20.0 g of polyethylene glycol (PEG-NH ₂ ) (molecular weight: 13900) with a methoxy group at one end and a 3-aminopropyl group at the other end was dissolved in 100 ml of N,N-dimethylformamide (DMF). To this solution was added 15.0 g of β-benzyl-L-aspartic acid-N-carboxylic anhydride (BLA-NCA). The polymerization was allowed to proceed for 24 hours while stirring in a 35°C water bath. Then, under stirring in an ice bath, the polymerization reaction solution was added to a 0.5N aqueous sodium hydroxide solution, and stirred for 20 minutes. The pH of the solution was then adjusted to approximately 4 by adding 2N hydrochloric acid, diluted with distilled water to a total volume of 20 liters, and then adjusted to pH 4 again. Next, the concentration and washing steps were repeated using a hollow fiber type ultrafiltration device (AmiconCH2, molecular weight cut-off value after ultrafiltration: 10000). Then, the resulting concentrated solution was purified with a sulfonic acid type ion exchange resin (Amberlite IR-120B) column. The resulting eluate was concentrated under reduced pressure, and then freeze-dried to obtain 19.58 g of polyethylene glycol-polyaspartic acid block copolymer (PEG-P(Asp.)). 5.008 g of PEG-P (Asp.) was dissolved in 83 g of DMF, and then 83 mL of acetonitrile was added thereto. To this solution was added 8.979 g of dicyclohexylcarbodiimide (DCC), stirred for 5 minutes, and then a solution prepared by dissolving 2.528 g of doxorubicin hydrochloride in 167 ml of DMF and adding 786 μl of triethylamine was added. It was then allowed to react for 4 hours with stirring at room temperature. After the reaction, 16.7 ml of 1% phosphoric acid aqueous solution was added and stirred for 5 minutes. After dialysis with a dialysis membrane (molecular weight cutoff = 12000-14000), the precipitate produced by DCC was removed by filtration. The resulting filtrate was purified with a hollow fiber type ultrafiltration device (Amicon CH2, ultrafiltration membrane molecular weight cutoff = 10000). This filtrate is further concentrated by ultrafiltration with ADVANTEC UK-50 ultrafiltration membrane (molecular weight cut-off value=50000), and obtaining 177 milliliters is 12 mg/ml (absorbance measured at 485 nanometers by ultraviolet spectrophotometer) by doxorubicin. Calculated) aqueous solution. The resulting PEG-P(Asp.) ADR has the structure shown in the above formula (2), wherein R ₁ is methyl, R ₂ is 1,3-propylene, R ₃ is methylene, and a part of R ₄ is Hydroxyl, the rest are residues of the above formula (3) (Y is CH ₂ OH, Z is H), R ₅ is hydrogen, n=315, m=30, x=8. The content of doxorubicin is 32.3% by weight, which has suitable water solubility. 20 milliliters containing 12 mg/ml (according to doxorubicin) obtained PEG-P (Asp.) ADR aqueous solution and by dissolving 258.8 milligrams of doxorubicin hydrochloride in 60 milliliters of DMF and adding 100 microliters of triethylamine to prepare The solution was mixed and the mixture was allowed to stir at room temperature in the dark for 2 hours. After dialysis with a dialysis membrane (molecular weight cut-off = 12000-14000), the resulting solution was freeze-dried. Purification and concentration were carried out by redissolving it in water and ultrafiltration with ADVANTEC UK-50 ultrafiltration membrane (molecular weight cut-off = 50000). After further filtration with a 0.45 micron filter, 25.3 ml of an aqueous solution of the block copolymer-drug complex preparation was obtained. The resulting aqueous solution was found to have the HPLC chromatogram shown in FIG. 13 . In this spectrum, peak ① is doxorubicin, peak ② is the dimer of doxorubicin, and broad peak ③ is doxorubicin linked to the polymer. The concentration of doxorubicin (peak ①) was 3.06 mg/ml, and the concentration of doxorubicin dimer (peak ②) was 3.18 mg/ml. The weight ratio of doxorubicin to doxorubicin dimer is 1:1.04.

The measurement conditions of HLPC are as follows.

Column: C4.300 Angstrom/5 micron, manufactured by Waters Co.;

Eluent: acetonitrile/1% acetic acid+40mM sodium dodecyl sulfate;

Gradient elution:

Time (min) 0 4 12 25 30 31

Acetonitrile concentration (%) 15 35 35 85 85 15

Detection: 485 nm

Flow rate: 1 ml/min.

Example 4

20 milliliters of the aqueous solution containing PEG-P (Asp.) ADR of 12 mg/ml (according to doxorubicin) prepared in embodiment 3 and the hydrochloride of dissolving 102.4 milligrams of doxorubicin in 60 milliliters of DMF and adding 32 The resulting solution of microliters of triethylamine was combined, and the mixture was stirred at room temperature in the dark for 2 hours. After dialysis with a dialysis membrane (molecular weight cut-off = 12,000-14,000), the resulting solution was freeze-dried. Purification and concentration were carried out by redissolving it in water and performing ultrafiltration with an ultrafiltration membrane ADVANTEC UK-50 (molecular weight cut-off = 50,000). It was further filtered with a 0.45 micron filter to obtain 20.4 ml of an aqueous solution of the block copolymer-drug composite preparation. The obtained aqueous solution was found to have the HPLC chromatogram shown in FIG. 14 . In the figure, peak 1 is doxorubicin, peak 2 is the dimer of doxorubicin, and broad peak 3 is doxorubicin linked to the polymer. The concentration of doxorubicin (peak 1) was 3.01 mg/ml and the concentration of doxorubicin dimer (peak 2) was 0.39 mg/ml. The weight ratio of doxorubicin to doxorubicin dimer is 1:0.13. The HPLC assay conditions are as described in Example 3.

Example 5

Dilute 20 ml of the aqueous solution of PEG-P(Asp.) ADR containing 12 mg/ml (as doxorubicin) prepared in Example 3 with 20 ml of water, then lyophilize. Dissolve in 20ml of water again, adjust the pH value of the solution with acetic acid and sodium acetate aqueous solution, make the solution finally become 40ml of 30mM acetic acid salt buffer solution (pH5.0). The prepared solution was mixed with 128.0 mg of doxorubicin hydrochloride and stirred at room temperature in the dark for 2 days. After dialysis with a dialysis membrane (molecular weight cut-off = 12,000-14,000), ultrafiltration was performed with an ultrafiltration membrane ADVANTEC UK-50 (molecular weight cut-off = 50,000) to purify and concentrate to obtain 16.7 ml of block copolymer-drug complex preparation of aqueous solution. The obtained solution was found to have the HPLC chromatogram shown in FIG. 15 . In the figure, peak 1 is doxorubicin, peak 2 is the dimer of doxorubicin, and broad peak 3 is doxorubicin linked to the polymer. The concentration of doxorubicin (peak 1) was 2.99 mg/ml and the concentration of doxorubicin dimer (peak 2) was 0.27 mg/ml. The weight ratio of doxorubicin to doxorubicin dimer is 1:0.09. The HPLC assay conditions are as described in Example 3. Application example 1

Colon 26 adenocarcinoma cells were implanted subcutaneously in the axilla of each CDF1 female mouse. When the tumor volume reaches about 100 mm ³ , the drug is administered intravenously once a day on the fourth day, three times in total, and the drug is the block copolymer-drug compound preparation synthesized in Example 3, 4 or 5, or doxorubicin hydrochloride Salt, (pointed out by the arrow in the figure), to examine the antitumor activity of the drug. Before use, each drug needs to be diluted with normal saline. The dose of each drug was determined according to the amount of peak 1 in the HPLC chromatogram of doxorubicin. The antitumor effect of each drug was evaluated according to the tumor growth curve, the number of mice with tumor disappearance, and the chemotherapy index. The results are shown in Tables 1 and 2, Figures 16 and 17. Compared with the case of doxorubicin hydrochloride administration, when the block copolymer-drug compound formulation of Example 3-5 was administered, it was observed that in a wider dose range, the mice with tumor disappearance There are only more. In particular, the agent with a higher content of doxorubicin dimer in Example 3 had the best results in terms of complete cure rate and chemotherapeutic index.

Table 1 Antitumor activity against mouse colon 26 adenocarcinoma sample Dose (mg/kg) tumor-free mice Doxorubicin Hydrochloride 510 0/52/5 The medicament of embodiment 3 2.5510 1/45/53/5 The medicament of embodiment 4 2.5510 0/51/54/5 The medicament of embodiment 5 2.5510 0/52/53/5

Table 2 Comparison of Chemotherapy Indexes sample _LD50 Min T/C ₄₂ ¹⁾ Chemotherapy index ²⁾ Doxorubicin Hydrochloride 15 5.95 2.52 The medicament of embodiment 3 15 2.59 5.78 The medicament of embodiment 4 15 4.88 3.08 The medicament of embodiment 5 15 6.02 2.49

1) After the initial drug administration, the minimum dose when T/C is 42% or less on day 14

2) Chemotherapy index: LD ₅₀ /(minimum T/C ₄₂ )

Example 6

Dissolve 20.0 g of polyethylene glycol (PEG-NH ₂ ) (molecular weight 14,200) with a methoxy group at one end and a 3-aminopropyl group at the other end in 100 mL of N,N-dimethylformamide (DMF) . 15.0 g of L-aspartic acid-β-benzyl ester-N-carboxylic acid anhydride (BLA-NCA) was added to the solution, followed by polymerization in a water bath at 35° C. under stirring for 24 hours. Then, under stirring in an ice bath, the polymer solution was added to a 0.5N aqueous sodium hydroxide solution, and stirred for 20 minutes. Add 2N hydrochloric acid to adjust the pH value to about 4, dilute the solution with distilled water to a total volume of 20 liters, and adjust the pH value to 4. The concentration and washing steps were repeated using a hollow fiber type ultrafiltration device (Amicon CH ₂ , molecular weight cutoff of ultrafiltration membrane = 10,000). Subsequently, the concentrated solution was purified with a sulfonic acid type ion exchange resin (Amberlite IR-120B) column. The resulting eluate was concentrated under reduced pressure and then freeze-dried to obtain 21.26 g of polyethylene glycol-polyaspartic acid block copolymer (PEG-P(Asp.)). 7.501 g of PEG-P(Asp.) was dissolved in 125 mL of DMF, followed by the addition of 125 mL of acetonitrile. Add 12.992 grams of dicyclohexylcarbodiimide (DDC) afterwards, after stirring for 5 minutes, add a solution, this solution is that 3.654 grams of adriamycin hydrochloride are dissolved in 250 milliliters of DMF, then add 1.14 milliliters of trimethylamine and preparation. Stir at room temperature and allow to react for 4 hours. After the reaction, 25 ml of 1% phosphoric acid aqueous solution was added and stirred for 5 minutes. After dialysis with a dialysis membrane (molecular weight cut-off value = 12,000-14,000), it was filtered to remove the precipitate generated by DCC. The obtained filtrate was purified with a hollow fiber type ultrafiltration device (Amicon CH ₂ , ultrafiltration membrane molecular weight cut-off=10,000), and then concentrated with an ultrafiltration membrane ADVANTEC UK-50 (molecular weight cut-off=50,000) to obtain 270 ml of 12 Aqueous solution of mg/ml doxorubicin (calculated from the absorbance at 485nm measured by UV spectrophotometer). The PEG-P(Asp.) ADR thus obtained has the structure of the above-mentioned formula (2), in which R ₁ is methyl, R ₂ is 1,3-propylene, R ₃ is A part of methylene, R ₄ is a hydroxyl group, and the rest is the residue of the above-mentioned formula (3) [Y is CH ₂ OH, Z is H], R ₅ is hydrogen, n=325, m=30 and x=8. The doxorubicin content was 32.4%, which showed adequate water solubility. Dissolve 1 ml of the thus obtained aqueous solution of PEG-P(Asp.) ADR containing 12 mg/ml (calculated as doxorubicin) and 11.93 mg of ¹⁴ C-labeled doxorubicin hydrochloride in 3 ml of DMF and add The resulting solution of 4.9 µl of triethylamine was mixed, and the mixture was stirred at room temperature in the dark for 2 hours. After dialysis with a dialysis membrane (molecular weight cut-off = 12,000-14,000), the resulting solution was purified and concentrated with an ultrafiltration membrane ADVANTEC UK-50 (molecular weight cut-off = 50,000) to obtain 3.0 ml of block copolymer-drug complex preparation of aqueous solution. The obtained solution was found to have the HPLC chromatogram shown in FIG. 18 . In the figure, peak 1 is doxorubicin, peak 2 is the dimer of doxorubicin, and broad peak 3 is doxorubicin linked to the polymer. The concentration of doxorubicin (peak 1) was 1.29 mg/ml and the concentration of doxorubicin dimer (peak 2) was 1.36 mg/ml. The weight ratio of doxorubicin to doxorubicin dimer is 1:1.05. The HPLC assay conditions are as described in Example 3.

Example 7

Dissolve 9.86 mg of ¹⁴ C-labeled adriamycin hydrochloride in 6.25 ml of DMF with 2.08 ml of PEG-P (Asp.) ADR solution prepared in Example 6 , and the solution formed by adding 3.3 μl of triethylamine was mixed, and the mixture was stirred at room temperature in the dark for 2 hours. After dialysis with a dialysis membrane (molecular weight cut-off = 12,000-14,000), purification and concentration were performed with an ultrafiltration membrane ADVANTEC UK-50 (molecular weight cut-off = 50,000) to obtain 2.3 ml of an aqueous solution of the block copolymer-drug composite preparation. The obtained aqueous solution was found to have the HPLC chromatogram shown in FIG. 19 . In the figure, peak 1 is doxorubicin, peak 2 is the dimer of doxorubicin, and broad peak 3 is doxorubicin linked to the polymer. The concentration of doxorubicin (peak 1) was 3.41 mg/ml and the concentration of doxorubicin dimer (peak 2) was 0.95 mg/ml. The weight ratio of doxorubicin to doxorubicin dimer is 1:0.28. The HPLC assay conditions are as described in Example 3.

Example 8

Dissolve 20.0 g of polyethylene glycol (PEG-NH ₂ ) (molecular weight 14,500) with a methoxy group at one end and a 3-aminopropyl group at the other end in 100 mL of N,N-dimethylformamide (DMF) . 15.0 g of L-aspartic acid-β-benzyl ester-N-carboxylic acid anhydride (BLA-NCA) was added to the solution, followed by polymerization in a water bath at 35° C. with stirring for 24 hours. Then, under stirring in an ice bath, the polymer solution was added to a 0.5N aqueous sodium hydroxide solution, and stirred for 20 minutes. 2N hydrochloric acid was added to adjust the pH to about 4, the solution was diluted with distilled water to a total volume of 20 liters, and then the pH was adjusted to 4. The steps of concentration and washing with water were repeated using a hollow fiber type ultrafiltration device (Amicon CH ₂ , molecular weight cutoff of ultrafiltration membrane = 10,000). Subsequently, the concentrated solution was purified with a sulfonic acid type ion exchange resin (Amberlite IR-120B) column. The resulting eluate was concentrated under reduced pressure and then freeze-dried to obtain 19.01 g of polyethylene glycol-polyaspartic acid block copolymer (PEG-P(Asp.)). 5.010 g of PEG-P(Asp.) was dissolved in 83 ml of DMF, and then 83 ml of acetonitrile was added thereto. Then mix with 8.693 grams of dicyclohexylcarbodiimide (DDC), and after stirring for 5 minutes, add a solution, which is to dissolve 2.445 grams of doxorubicin hydrochloride in 167 milliliters of DMF, and add 759 microliters of trimethyl Amine preparation. Thereafter, stirring was carried out at room temperature to allow a reaction for 4 hours. After the reaction, 16.7 ml of 0.5% phosphoric acid aqueous solution was added and stirred for 5 minutes. After dialysis with a dialysis membrane (molecular weight cut-off = 12,000-14,000), the precipitate formed by DCC was removed by filtration. The resulting filtrate was concentrated with an ultrafiltration membrane ADVANTEC UK-50 (molecular weight cut-off=50,000) to obtain 185 ml of an aqueous solution having a concentration of 12 mg/ml in terms of doxorubicin (calculated from the absorbance at 485 nm measured by a UV spectrophotometer). The PEG-P(Asp.) ADR thus obtained has the structure of the above-mentioned formula (2), in which R ₁ is methyl, R ₂ is 1,3-propylene, R ₃ is propylene Methyl, part of _R4 is hydroxyl, the rest is the residue of formula (3) mentioned above [Y is _CH2OH , Z is H], _R5 is hydrogen, n=350, m=32 and x =8. The doxorubicin content was 30.2%, which showed suitable water solubility. 20 ml of an aqueous solution of PEG-P(Asp.) ADR thus obtained containing 12 mg/ml (calculated as doxorubicin) was dissolved in 60 ml of DMF with 564.0 mg of doxorubicin hydrochloride and added 176 μg The resulting solution of triethylamine was mixed, and the mixture was stirred at room temperature in the dark for 2 hours. After dialysis with a dialysis membrane (molecular weight cut-off = 12,000-14,000), the resulting solution was freeze-dried. The resulting solution was purified and concentrated by redissolving it in water, and using an ultrafiltration membrane ADVANTEC UK-50 (molecular weight cut-off = 50,000). It was further filtered with a 0.45 micron filter to obtain 59.4 ml of an aqueous solution of the block copolymer-drug composite preparation. The obtained aqueous solution was found to have the HPLC chromatogram shown in FIG. 20 . In the figure, peak 1 is doxorubicin, peak 2 is the dimer of doxorubicin, and broad peak 3 is doxorubicin linked to the polymer. The concentration of doxorubicin (peak 1) was 1.10 mg/ml and the concentration of doxorubicin dimer (peak 2) was 3.07 mg/ml. The weight ratio of doxorubicin to doxorubicin dimer is 1:2.79. The HPLC assay conditions are as described in Example 3.

Example 9

Dissolve 32.0 mg of adriamycin hydrochloride in 30 ml of DMF with 10 ml of aqueous solution of PEG-P (Asp.) ADR prepared in Example 8 and add 10.0 micrograms of The resulting solution of triethylamine was mixed, and the mixture was stirred at room temperature in the dark for 2 hours. After dialysis with a dialysis membrane (molecular weight cut-off = 12,000-14,000), the resulting solution was freeze-dried. Redissolved in water, purified and concentrated with ultrafiltration membrane ADVANTEC UK-50 (molecular weight cut-off = 50,000). It was further filtered with a 0.45 micron filter to obtain 9.1 ml of an aqueous solution of the block copolymer-drug composite preparation. The obtained aqueous solution was found to have the HPLC chromatogram shown in FIG. 21 . In the figure, peak 1 is doxorubicin, peak 2 is the dimer of doxorubicin, and broad peak 3 is doxorubicin linked to the polymer. The concentration of doxorubicin (peak 1) was 1.98 mg/ml and the concentration of doxorubicin dimer (peak 2) was 0.12 mg/ml. The weight ratio of doxorubicin to doxorubicin dimer is 1:0.06. The HPLC assay conditions are as described in Example 3.

Example 10

Dissolve 51.4 mg of adriamycin hydrochloride in 15 ml of DMF with 5 ml of aqueous solution of PEG-P (Asp.) ADR prepared in Example 8 and add 16.0 micrograms of The resulting solution of triethylamine was mixed, and the mixture was stirred at room temperature in the dark for 2 hours. After dialysis with a dialysis membrane (molecular weight cut-off = 12,000-14,000), the resulting solution was freeze-dried. Redissolved in water, purified and concentrated with ultrafiltration membrane ADVANTEC UK-50 (molecular weight cut-off = 50,000). It was further filtered with a 0.45 micron filter to obtain 15.2 ml of an aqueous solution of the block copolymer-drug composite preparation. The obtained aqueous solution was found to have the HPLC chromatogram shown in FIG. 22 . In the figure, peak 1 is doxorubicin, peak 2 is the dimer of doxorubicin, and broad peak 3 is doxorubicin linked to the polymer. The concentration of doxorubicin (peak 1) was 1.04 mg/ml and the concentration of doxorubicin dimer (peak 2) was 0.88 mg/ml. The weight ratio of doxorubicin to doxorubicin dimer is 1:0.85. The HPLC assay conditions are as described in Example 3.

Example 11

In 65 milliliters of DMF, dissolve 103.5 milligrams of the doxorubicin dimer and 49.1 milligrams of doxorubicin hydrochlorides prepared by embodiment 1 (2), the obtained solution and the preparation of embodiment 8 contain 12 mg/ml (according to doxorubicin 23 ml of an aqueous solution of PEG-P(Asp.) ADR was mixed, and the mixture was stirred at room temperature in the dark for 2 hours. After dialysis with a dialysis membrane (molecular weight cutoff = 12,000-14,000), purification and concentration were performed with an ultrafiltration membrane ADVANTEC UK-50 (molecular weight cutoff = 50,000). It was further filtered with a 0.45-micron filter to obtain 14.9 ml of an aqueous solution of the block copolymer-drug composite preparation. The obtained aqueous solution was found to have the HPLC chromatogram shown in FIG. 23 . In the figure, peak 1 is doxorubicin, peak 2 is the dimer of doxorubicin, and broad peak 3 is doxorubicin linked to the polymer. The concentration of doxorubicin (peak 1) was 1.13 mg/ml and the concentration of doxorubicin dimer (peak 2) was 2.76 mg/ml. The weight ratio of doxorubicin to doxorubicin dimer is 1:2.44. The HPLC assay conditions are as described in Example 3.

Example 12

1.0034 g of PEG-P (Asp.) prepared in Example 8 was dissolved in 16.7 ml of DMF, and then 16.7 ml of acetonitrile was added thereto. Add 1.7504 g of dicyclohexylcarbodiimide (DDC), stir for 5 minutes, and then mix with a solution prepared by dissolving 474.4 mg of daunorubicin hydrochloride in 33.3 ml of DMF and adding 152 μl of triethylamine. React at room temperature for 4 hours. After the reaction, 3.3 ml of 0.5% phosphoric acid aqueous solution was added and stirred for 5 minutes. After dialysis with a dialysis membrane (molecular weight cut-off = 12,000-14,000), the precipitate generated by DCC was removed by filtration. Concentrate the filtrate produced with ultrafiltration membrane ADVANTEC UK-50 (molecular weight cut-off=50,000), and obtain 36 milliliters of aqueous solutions containing daunorubicin at a concentration of 12 mg/ml (according to the absorbance calculation at 485nm measured by a UV spectrophotometer) . The PEG-P(Asp.) DAM thus obtained has the structure of the above-mentioned formula (2), in which R ₁ is methyl, R ₂ is 1,3-propylene, R ₃ is propylene A part of methyl, R ₄ is a hydroxyl group, and the rest is the residue of the above-mentioned formula (3) [Y is CH ₃ , Z is H], R ₅ is hydrogen, n=350, m=32 and x= 8. The daunorubicin content was 30.3%, which showed suitable water solubility. 7 milliliters of the aqueous solution of PEG-P (Asp.) DAM thus obtained containing 12 mg/ml (calculated as daunorubicin) was dissolved in 21 milliliters of DMF with 179.8 milligrams of doxorubicin hydrochloride and added 55.9 micrograms The resulting solution of triethylamine was mixed, and the mixture was stirred at room temperature in the dark for 2 hours. After dialysis with a dialysis membrane (molecular weight cut-off = 12,000-14,000), the resulting solution was freeze-dried. It was redissolved in water, purified and concentrated with an ultrafiltration membrane ADVANTEC UK-50 (molecular weight cut-off=50,000), and then further filtered with a 0.45 micron filter to obtain 16.5 ml of an aqueous solution of the block copolymer-drug composite preparation. The obtained aqueous solution was found to have the HPLC chromatogram shown in FIG. 24 . In the figure, peak 1 is doxorubicin, peak 2 is the dimer of doxorubicin, and broad peak 3 is daunorubicin attached to the polymer. The concentration of doxorubicin (peak 1) was 1.07 mg/ml and the concentration of doxorubicin dimer (peak 2) was 3.26 mg/ml. The weight ratio of doxorubicin to doxorubicin dimer is 1:3.05. The HPLC assay conditions are as described in Example 3. Application example 2

Colon 26 adenocarcinoma cells were subcutaneously transplanted into the axilla of each CDF1 female mouse, and after the transplantation, the mice were administered intravenously with the drug for 12 days, and the drug was the block copolymer-drug compound preparation prepared in Example 6 or 7, or ¹⁴ C-labeled doxorubicin hydrochloride. Each drug was diluted with normal saline before use. Blood samples were collected 15 minutes, 1, 4, 24 and 48 hours after dosing, and various organs were excised. Drug concentrations in plasma and in each internal organ were determined by measuring radioactivity with a liquid scintillation counter. In this assay, both doxorubicin and doxorubicin dimer are labeled with ¹⁴ C. When the total amount of doxorubicin and doxorubicin dimer in the given pharmaceutical product is set as 100, the periodic change of doxorubicin and doxorubicin dimer total amount in 1 milliliter plasma (% dose/ml ) are listed in Table 3. When only doxorubicin hydrochloride was administered, the drug in the plasma disappeared rapidly after administration, while in the case of using the medicament of the present invention, the drug in the plasma remained at a higher level for a longer period of time. The improvement in drug retention was most pronounced for the formulation of Example 6 containing higher doxorubicin dimer. When the total amount of doxorubicin and doxorubicin dimer in the given pharmaceutical preparation is set as 100, the periodic change of doxorubicin and doxorubicin dimer total amount (% dose/ grams) are listed in Table 4. Table 5 lists the cyclic variation (% dose/gram) of the above total amounts in the heart. Compared with the case of administration of doxorubicin hydrochloride alone, when the agent of the present invention was used as the drug administered, its initial concentration was slightly lower, and decreased over time in the heart, but in the tumor area, the drug was several times higher. Concentrations accumulate and increase over time. The agent of Example 6 containing a higher level of doxorubicin dimer has the most obvious drug accumulation in the tumor. Application example 3

Colon 26 adenocarcinoma cells were implanted subcutaneously in the axilla of each CDF1 female mouse. When the tumor volume reached about 100 mm , the drug was given intravenously once every day on the fourth day ^, three times in total, and the drug was the block copolymer-medicine composite preparation prepared in Example 8 or 12, or doxorubicin hydrochloride ( Pointed out by the arrow in the figure), to examine the antitumor activity of the drug. For the drug of Example 8, the effect of only the first administration was also examined. Before use, each drug needs to be diluted with normal saline. The dose of each drug was determined according to the amount of doxorubicin in peak 1 in the HPLC chromatogram. According to the tumor growth curve and the number of mice with tumor disappearance, the antitumor effect of each drug was evaluated. The results are presented in Table 6, Figures 25, 26 and 27. Compared with the case of doxorubicin hydrochloride administration, when the pharmaceutical agent of Example 8 or 12 was administered, a larger number of mice with tumor disappearance was observed in a wider dose range. Application example 4

Colon 26 adenocarcinoma cells were subcutaneously transplanted into the axilla of each CDF1 female mouse. After transplantation, the mice were administered intravenously for 8 days, and the drug was the block copolymer-drug compound preparation prepared in Example 8 or 9. Each drug was diluted with normal saline before use. Blood samples were collected 15 minutes and 1, 4, 24 and 48 hours after dosing, and various organs were excised. Concentrations of doxorubicin and doxorubicin dimer in plasma and tumors were determined by extracting the drug with an organic solvent and measuring it by HPLC. When the amount of doxorubicin in the given pharmaceutical preparation is set as 100, the periodic change (% dose/ml) of doxorubicin in 1 ml of plasma, and when the amount of doxorubicin dimer in the given pharmaceutical preparation is set as At 100, the periodic variation (% dose/ml) of the amount of doxorubicin dimer in 1 ml of plasma is listed in Table 7. The improvement in drug retention in blood was most pronounced for the formulation of Example 8 containing higher amounts of doxorubicin dimer. When the amount of doxorubicin in the given pharmaceutical preparation is set as 100, the periodic change of the amount of doxorubicin in 1 gram of tumor tissue (% dose/gram), and when the amount of doxorubicin dimer in the given drug When it is set as 100, the cycle change (% dose/gram) of the amount of doxorubicin dimer in 1 gram of tumor tissue is listed in Table 8. The agent of Example 8 containing a higher level of doxorubicin dimer had the most significant improvement in the accumulation of the drug in the tumor. Application example 5

Colon 26 adenocarcinoma cells were subcutaneously transplanted into the underarm of each CDF1 female mouse, and after transplantation, the mice were administered intravenously for 11 days, and the drug was the block copolymer-drug compound preparation prepared in Example 8, 10 or 12 . Each drug was diluted with normal saline before use. One or 24 hours after dosing, blood samples were collected and various organs were excised. Concentrations of doxorubicin and doxorubicin dimer in plasma and tumors were determined by extracting the drug with an organic solvent and measuring it by HPLC. When the amount of doxorubicin in the given pharmaceutical preparation is set as 100, the periodic change of doxorubicin in 1 ml of plasma (% dose/ml), and when the amount of doxorubicin dimer in the given pharmaceutical preparation is set as 100 Table 9 shows the periodic changes (% dose/ml) of the amount of doxorubicin dimer in 1 ml of plasma. The results obtained with the agent of Example 8 were almost the same as those obtained using Example 4. Since the ratio of doxorubicin dimer in the medicament of Example 10 is lower than that of the medicament of Example 8, its retention value in blood is slightly lower. When the amount of doxorubicin in the given pharmaceutical preparation is set as 100, the periodic change of the amount of doxorubicin in 1 gram of tumor tissue (% dose/gram), and when the amount of doxorubicin dimer in the given drug When it is set as 100, the cycle change (% dose/gram) of the amount of doxorubicin dimer in 1 gram of tumor tissue is listed in Table 10. The results obtained with the drug of Example 8 were almost the same as those obtained in Application Example 4. Since the ratio of doxorubicin dimer in the medicament of Example 10 is lower than that of the medicament of Example 8, its accumulation value in the tumor is also slightly lower.

Table 3 Cycle change (% dose/ml) (plasma) sample time after administration 15 minutes 1 hour 4 hours 24 hours 48 hours (A)(B)(C) 0.4761.641.7 0.2953.729.2 0.3639.221.3 0.1015.98.2 0.074.72.2 (A) doxorubicin hydrochloride (B) the medicament of embodiment 6 (C) the medicament of embodiment 7

Table 4 Cycle change (% dose/gram) (tumor) sample time after administration 15 minutes 1 hour 4 hours 24 hours 48 hours (A)(B)(C) 2.32.32.3 2.43.82.7 1.74.94.9 1.39.67.0 1.09.14.0 (A) doxorubicin hydrochloride (B) the medicament of embodiment 6 (C) the medicament of embodiment 7

Table 5 Cycle change (% dose/gram) (cardiac) sample time after administration 15 minutes 1 hour 4 hours 24 hours 48 hours (A)(B)(C) 10.54.96.5 7.04.25.4 4.63.33.8 1.11.61.4 0.51.00.7 (A) doxorubicin hydrochloride (B) the medicament of embodiment 6 (C) the medicament of embodiment 7

Table 6 Antitumor activity against mouse colon 26 adenocarcinoma sample Dose (mg/kg) tumor-free mice Doxorubicin Hydrochloride 510 0/52/5 The medicament of embodiment 8 510 ^* 5/55/5 The medicament of embodiment 12 1.252.5510 0/50/54/55/5

( ^* first dose only)

Table 7 Cycle change (% dose/ml) (plasma) sample time after administration 15 minutes 1 hour 4 hours 24 hours 48 hours (A)(1)(2) 44.967.2 32.969.7 20.761.9 3.814.2 2.03.9 (B)(1)(2) 27.723.0 12.322.5 5.319.4 1.911.1 0.57.0 (A) the medicament of embodiment 8 (B) the medicament of embodiment 9 (1) doxorubicin (2) doxorubicin dimer

Table 8 Cycle change (% dose/gram) (tumor) sample time after administration 15 minutes 1 hour 4 hours 24 hours 48 hours (A)(1)(2) 4.01.1 3.11.0 6.86.4 19.014.1 20.412.2 (B)(1)(2) 1.30.0 2.18.1 1.92.8 2.23.9 1.24.9 (A) the medicament of embodiment 8 (B) the medicament of embodiment 9 (1) doxorubicin (2) doxorubicin dimer

Table 9 Cycle change (% dose/ml) (plasma) sample time after administration 1 hour 24 hours (A)(1)(2) 36.783.5 6.817.9 (B)(1)(2) 11.065.3 4.116.2 (C)(1)(2) 40.6100.2 10.224.5 (A) The medicament of Example 8 (B) The medicament of Example 10 (C) The medicament of Example 12 (1) Adriamycin (2) Adriamycin dimer

Table 10 Cycle change (% dose/gram) (tumor) sample time after administration 1 hour 24 hours (A)(1)(2) 3.41.8 21.016.1 (B)(1)(2) 2.60.1 4.53.0 (C)(1)(2) 5.02.1 9.16.3 (A) The medicament of Example 8 (B) The medicament of Example 10 (C) The medicament of Example 12 (1) Adriamycin (2) Adriamycin dimer

By adding anticancer drugs and the like to the inner core of the micelle of the block copolymer, endow the polymer block copolymer-drug compound preparation containing two, three, and four polymers of the present invention with higher drug efficacy and lower toxicity. The present invention thus provides remarkably effective pharmaceutical formulations.

Claims (2)

1. A doxorubicin dimer having the following structural formula (AA):

2. The trimer of doxorubicin is characterized in that it is prepared by directly chemically combining doxorubicin molecules or their acid salts with each other, and has the mass spectrum shown in FIG. 7 .

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